Molecular mechanisms and functional implications of Ded1p phaseseparation.

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Ded1p (DDX3) is an RNA DEAD box helicase that removes secondary structure elements in the 5’ UTR (untranslated region) of an mRNA. This facilitates the scanning of the 40S ribosomal subunit such that the translation initiation codon can be identified. Upon cellular stress, Ded1p is recruited to cytoplasmic stress granules that contain mRNPs (messenger ribonucleoproteins) that are stalled in translation. Functionally, stress granule formation protects the embedded mRNAs against degradation and induces changes in gene expression that up-regulate stress response. Here, we address the molecular details behind the interactions that are responsible for Ded1p condensation into stress granules. In addition, we address how Ded1p activity is modulated by this condensation process. Experimentally, we will make use of biochemical methods to determine which Ded1p amino-acids contribute to the LLPS (liquid-liquid phase separation) propensity of the enzyme. Based on biophysical studies, in particular NMR spectroscopy, we will reveal which transient intermolecular contacts take place upon condensation and how the dynamics of the protein is affected by these interactions. These data will be combined with computational methods to predict and visualize the LLPS behavior of Ded1p. To address how Ded1p helicase activity is modulated by LLPS we will determine the turnover rates of Ded1p varients that show altered condensation propensities. Based on a large number of these mutated proteins we aim to establish a direct correlation between activity and phase separation. These structural and catalytic studies will be complemented with experiment in the presence of (capped) RNA and components of the mRNA cap binding complex. Together, our data will thereby contribute to the understanding of how Ded1p activity is regulated through stress granule formation. Our in vitro insights into Ded1p condensation, interactions and activity will be complemented with experiments that address the relevance of our findings in vivo.In summary, our work will provide mechanistical insights into the atomic details that result in Ded1p LLPS and reveal how LLPS effects catalytic activity and cellular function. Furthermore, our results will enhance the general understanding of the molecular basis of LLPS.